Both types of human 5a-reductases are hydrophobic, microsomal enzymes with a molecular mass of approximately 29 kDa (Table 1). Type 1 isoenzyme, composed of 259 amino acids, has an optimal pH of 6.5 to 8.0, whereas type 2 isoenzyme is composed of 254 amino acids and has an optimal pH of 5.5 (Fig. 2). The determi- nants of androgen binding are encoded at the ends of the molecule, while those of NADPH binding are encoded in the carboxyl terminal half of the enzyme. Both isoenzymes have similar substrate preferences for gene structures (9). An average of 37% of the amino acids have side chains commonly found buried in the hydrophobic interior of globular proteins. These hydrophobic residues are dis- tributed throughout the enzyme and do not give rise to clear-cut transmembrane regions in hydropathy plots. This structural feature suggests the 5a-reductase iso- enzymes are intrinsic membrane proteins deeply embedded in the lipid bilayer (15).
The enzymes have never been fully biochemically characterized (11), as the isolation and purification have been difficult due to the insolubility and, generally, the rather low activity of these membrane-bound proteins. Since X-ray patterns are still to be determined (16), only indirect information is available on the
TABLE 1 Properties of Human Type 1 and Type 2 5a-Reductases
FIGURE 2 Activities of human recombinant type 1 and type 2 5a-reductase expressed in CHO cells. The activities were determined over the indicated pH range in the presence of 1 to 21.5 mM [14C] testosterone and have been normalized to the highest value in each data set. They consequently are represented as relative enzyme activities. O ¼ type 1 isoenzyme (50 pmol min21 mg21), B ¼ type 2 isoenzyme (15 pmol min21 mg21). Source: Adapted from Ref. 19.
three-dimensional structures of their binding sites. To circumvent these difficulties, expression-cloning strategies have been used to isolate cDNA (17).
Genetically, the type 1 isoenzyme is encoded by the SRD5A1 gene on the short arm of chromosome 5 (band p15), while the type 2 isoenzyme by the SRD5A2 gene on chromosome 2 (band p23). Both gene structures are similar in that each contains five exons separated by four introns (14).
Western blot studies of human cell cultures (sebocytes, keratinocytes, fibro- blasts, dermal microvascular endothelial cells, and melanocytes) by Chen et al. (8) show two closely lying bands of type 1 5a-reductase in the range of 21 to
27 kDa, possibly indicating the existence of heterogeneous proteins. This finding is supported by a study of Lopez-Solache (18) that reveals the presence of two forms of mRNA species for rat type 1 5a-reductase. Possible explanations of the two bands could be:
1. partial protein denaturation during the process of extraction or isolation,
2. the existence of the enzyme in two states, activated or inactivated, through either phosphorylation, dephosphorylation (8), or proteolytic cleavage of a zymogen, and
3. alternative splicing of mRNA.
Intraspecies and interspecies comparisons of the amino acid sequences between type 1 and type 2 isoenzymes show only moderate homologies, suggesting differing structure, functions, and/or regulations. Homology between the human type 1 and type 2 5a-reductases is approximately 50% (Fig. 3). The type 1 human and rat iso- enzymes have approximately 61% amino acid sequence identity, while the two cor- responding type 2 5a-reductases are 75% homologous (Table 1). The considerable differences of the enzymes concerning protein structure, functional characteristics,
FIGURE 3 Amino acid sequences of human type 1 and type 2 5a-reductase. Shaded amino acids represent homologous items. Asterisks above the sequence stand for amino acid positions of naturally occurring mutants in type 2 isoenzyme deficient patients. Hash keys above the sequence denote the tetrapeptide sequence implicated in Finasteride binding. Source: Adapted from Ref. 19.
and tissue distribution would seem to make the rat a poor choice as a model for comparative in vivo pharmacological assessment of novel human 5a-reductase inhibitors. For example, rat type 1 isoenzyme is at least 50-fold more susceptible to inhibition by Finasteride than its human counterpart and the activities of both type 1 and type 2 5a-reductases can be monitored in rat prostate, while activity of the type 2 isoenzyme predominates in tissue from the mature human organ (19).
In the Cynomolgus monkey, 5a-reductases are highly homologous to their human protein analogs, sharing about 93% (type 1) and 95% (type 2) amino acid sequence identity. The results of inhibition studies indicate that the monkey isoenzymes are comparable on a molecular level to their respective human counterparts, supporting the relevance and use of the Cynomolgus monkey as a pharmacological model for in vivo evaluation of 5a-reductase inhibitors (19).
Enzymatic Activity and Specificity
5a-reductase, or more precisely NADPH: 4-ene-3-oxosteroid 5-oxidoreductase (EC
220.127.116.11), is a NADPH-dependent enzyme that selectively and irreversibly catalyzes the reduction of the 4,5-double bond of 4-ene-3-oxosteroids (e.g., testosterone,
FIGURE 4 (A) Proposed mechanism of the catalysis of testosterone to DHT by 5a-reductase. (B) Substrate-like transition state (Left) and product-like transition state (Right). Source: Adapted from Ref. 16.
progesterone, and androstenedione) into the corresponding 5a-3-oxosteroids (DHT,
5a-dihydroprogesterone, and 5a-androstanedione) (13).
The proposed mechanism (Fig. 4A) of testosterone reduction to DHT by
5a-reductase catalysis involves, as a key step, the activation of the 4-en-3-on moiety of testosterone by the interaction of the carbonyl group with an electrophilic residue Eþ of the active site, followed by hydride transfer from NADPH to the pos- ition 5 (16). This leads to enolate formation at C-3, C-4, which presumably is stabil- ized by Eþ at the active site. The process may be viewed alternatively as activation of the enone by Eþ leading to a positively polarized species, which accepts a hydride from NADPH at C-5. Enzyme mediated tautomerism then leads to DHT with release of NADPþ (5). Thus, it is possible to postulate two different transition states: the “substrate-like” transition state in which the C-5 has not yet changed its sp2 hybridization and the “product-like” transition state, in which the C-5 has assumed the final sp3 hybridization (Fig. 4B) (16).
The differences in primary sequences result in unique functional character- istics of the two isoenzymes. It has been proposed that the two isoenzymes have different roles in androgen metabolism (13).
5a-reductases have apparent Km values in the micro- to nanomolar range for steroid substrates (Table 2) (20). The relative affinity of D4(5)-steroids for type 1 iso- enzyme is:
Note: Type 1 and type 2 5a-reductase activities were determined at pH 7.5 (50 mM sodium phosphate) and pH 5.0 (50 mM sodium citrate), respectively. 1 mM glucose-6-phosphate and 0.5 U/mL glucose-6-phosphate dehydrogenase was included as a cofactor regenerating system (NADPþ to NADPH). Experiments were conducted using recombinant type 1 (0.05 – 0.10 mg protein per assay) and type 2 (0.02 – 0.15 mg protein per assay) isoenzymes expressed in CHO cells. Standard errors for all values were less than 20% of the indicated entries.
Source: Adapted from Ref. 19.
and for the type 2 isoenzyme:
progesterone ! 20a hydroxyprogesterone ! testosterone
! androstenedione ! corticosterone
The conversion of testosterone to DHT amplifies the androgenic signal through four mechanisms:
1. DHT, unlike testosterone, cannot be aromatized to estrogen, thus its effect remains purely androgenic (14).
2. DHT binds to the human androgen receptor with a several-fold higher affinity than testosterone does (14). It has also been suggested that the DHT/androgen receptor complex has a higher affinity for the acceptor site in the nuclear chromatin (5).
3. The DHT/androgen receptor complex appears to be more stable (14).
4. Both the 5a-reductase genes are adjusted by DHT via a feed-forward regulation (13).
Although testosterone and DHT share the same androgen receptor, it is not sur- prising that the two androgens exert varying physiological effects.
Localization, Distribution, and Cutaneous Enzymatic Activity
The specific localization and activity of type 1 isoenzyme in sebaceous glands makes this isoenzyme a potential therapeutic target for the treatment of acne. For this reason, it becomes important to understand where and how the two isoen- zymes of 5a-reductase act in the skin (21).
Type 1 5a-reductase is present, above all, in the skin, whereas type 2 is predo- minantly located in the prostate and in genital skin (9). Both the isoenzymes are expressed in the liver. In humans type 1 is not detectable in the fetus but is perma- nently expressed in skin and scalp from the time of puberty (13).